High-performance liquid chromatography of amino acids, peptides and proteins. XCVII. The influence of the gradient elution mode and displacer salt type on the retention properties of closely related protein variants separated by high-performance anion-exchange chromatography

The influence of different elution modes, gradient times and flow-rates on the relative retention of closely related variants of carbonic anhydrase and ovalbumin has been investigated using high-performance ion-exchange chromatography. Three isoform species of carbonic anhydrase and four isoforms related to ovalbumin eluted by anion-exchange chromatography were characterised by isoelectric focusing and sodium dodecylsulphate-polyacrylamide electrophoresis. Gradient retention data were collected using several different alkali metal halides as the displacer salt, in order to systematically evaluate the efffect on selectivity of different anions and cations in the series F^-, Cl^- and Br^-, and Li⁺, Na⁺ and K⁺. While the selectivity between the different ovalbumin isoform species remained essentially constant with each displacer salt, solute Z_c-values [J. Chromatogr., 458 (1988) 27] varied with the type of salt. In contrast, non-parallel retention plots were obtained for the carbonic anhydrase isoforms with the Z_c values different for each isoform. Furthermore, significant differences in chromatographic behaviour for these proteins were observed between experiments carried out under gradient elution conditions with either varied gradient time and constant flow-rates or fixed gradient time and varied flow-rates. These results are discussed in tems of the influence of column residence time and protein-salt interactions of the solute's interactive ionotope and the concomitant effects these structural perturbations may have on chromatographic behaviour.